Treatment of heart failure with growth hormone

ABSTRACT

Heart failure following myocardial infarction is treated by administration of an angiotensin II inhibitor for 8-12 weeks, followed by administration of a growth hormone for 1-3 weeks.

CROSS REFERENCE

[0001] This application is a continuation application of U.S. Ser. No.08/869,237, filed Jun. 4, 1997, which is incorporated herein byreference in its entirety and to which application we claim priorityunder 35 USC § 120.

BACKGROUND OF THE INVENTION

[0002] Heart failure occurs in three to four million individualsannually in the United States, and is a highly important cause ofcardiac morbidity and mortality. In about 60% of the patients, the heartfailure is secondary to late stage coronary disease, and in most of theremainder it is due to primary myocardial disease in the form ofidiopathic dilated cardiomyopathy. Treatment for severe heart failurehas improved with the addition of angiotensin converting enzyme (ACE)inhibitors to standard therapy, but despite such treatment the outlookremains poor in symptomatic patients (mortality about 10% per year), andlimiting cardiac symptoms often persist. Cardiac transplantation is adefinitive therapy for severe heart failure in some individuals, butthere is a need for new adjunctive medical therapies to improvefunctional status and provide a more favorable prognosis. Previousfindings in animal models, and a recent study in patients with heartfailure, showing favorable effects of growth hormone (GH) suggest thatGH treatment may offer such an adjunctive measure. Bunting et al.,WO95/28173 disclosed treatment of congestive heart failure byadministration of GH. Clark et al., U.S. Pat. No. 5,610,134 disclosedtreatment of congestive heart failure by administration of GH andinsulin-like growth factor 1 (“IGF1”), with or without an angiotensin IIconverting enzyme (ACE) inhibitor. However, our studies in animals withheart failure have shown that an ACE inhibitor in high dose may diminishthe beneficial effects of GH, including its action to promote aphysiologic form of hypertrophy.

SUMMARY OF THE INVENTION

[0003] We have now invented a new method for treating heart failurewhich follows myocardial infarction by administering an angiotensin II(AT₁) receptor blocker for 8-12 weeks, followed thereafter byadministration of a growth hormone for 1-3 weeks.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] The term “cardiac ischaemia” as used herein refers to theinterruption of oxygen supply to the cardiac muscle (which may be acuteor chronic). Cardiac ischaemia caused by obstruction can lead tomyocardial infarction. A large myocardial infarction, in turn, can leadto heart failure, a condition in which the function of the leftventricle is impaired and inadequate to meet the body's needs at rest orduring stress. Heart failure can be due to other functions as well, orof unknown etiology (idiopathic).

[0005] The term “angiotensin II inhibitor” refers to a compound capableof inhibiting or reducing the activity or effect of angiotensin II (theactive form of angiotensin). Angiotensin II inhibitors include compoundswhich bind, inhibit, or compete for AT₁ receptors (described by Fujisawaet al., U.S. Pat. No. 5,595,882, incorporated herein by reference). Anexemplary, presently preferred AT₁ inhibitor is losartan. Other AT₁inhibitors may be identified by determining their binding affinity tothe AT₁ receptor as set forth in U.S. Pat. No. 5,595,882.

[0006] The term “ventricular remodeling” refers to the alteration inchamber size, wall thickness, and other dimensional changes which occurin response to myocardial damage.

[0007] The term “growth hormone” in general refers to a protein orpolypeptide, whether natural, recombinant, or chemical in origin, whichis capable of stimulating the growth or proliferation of normal cellsunder appropriate conditions. “GH” and “hGH” refer to human growthhormone and its variations, including Protropin® (Genentech Inc.),Nutropin® (Genentech, Inc.), placental GH (U.S. Pat. No. 4,670,393), andthe variants described in WO90/04788 and WO92/09690, all incorporatedherein by reference. GH may also be modified, for example by conjugationwith polyoxyethylene (“PEG”) to form “PEGylated GH” as described in'134. Another growth factor which is endogenous and mediates some of theeffects of growth hormone is insulin-like growth factor-1 (IGF- 1).IGF-1 has been administered as an alternative for growth hormone intreating rats with heart failure, as described by R. Duerr et al., JClin Invest (1995) 95:619-27. IGF-1 may be used in conjunction with orin lieu of GH in the practice of the invention.

[0008] The terms “treating” and “treatment” as used herein refer toreduction in severity and/or frequency of symptoms, elimination ofsymptoms and/or underlying cause, prevention of the occurrence ofsymptoms and/or their underlying cause, and improvement or remediationof damage.

[0009] The term “subject” as used herein refers to a mammal,particularly a mammal having heart failure, preferably a human.

[0010] We have now developed a method for maximizing the effect of GHtreatment for heart failure following myocardial infarction. AngiotensinII effects were inhibited for 2½ months after myocardial infarctionusing a specific blocker of the angiotensin type 1 (AT₁) receptor whichis known to inhibit post-infarction hypertrophy and unfavorableremodeling of the left ventricle (LV), reduce myocardial fibrosis, andimprove the cardiac output in this experimental model of heart failure.Such effects occurred in the present study and effects of remodelingafter 2½ months of AT₁ treatment alone were detected at 2 weeks afterthe AT₁ blocker was discontinued compared to untreated animals. Inanother group of rats, following AT₁ receptor blockade with losartan for2½ months after myocardial infarction, GH was then given alone for 2weeks. In this setting, GH significantly improved myocardialcontractility and LV performance, increased LV muscle mass and wallthickness, enhanced LV diastolic function, and increased the cardiacoutput and cardiac index (collectively “favorable remodeling”).“Favorable remodeling” is an improvement in any of the above-mentionedfunctions or parameters of at least 20% over post-infarction levels,preferably at least 25% or greater. Thus, following acute myocardialinfarction an initial sustained inhibition of the unfavorable effects ofangiotensin II on remodeling is followed by GH, in a sequential manner,and constitutes a new treatment for heart failure. This sequentialtreatment may be repeated cyclicly if desired.

[0011] Suitable angiotensin II inhibitors will reduce or eliminateunfavorable remodeling effects. Presently preferred angiotensin IIinhibitors are AT₁ receptor blockers, for example losartan. Suitablegrowth hormones will improve cardiac function, improving one or more ofmyocardial contractility, LV performance, LV muscle mass, LV wallthickness, LV diastolic function, cardiac output and cardiac index. Thepresently preferred growth hormone is human growth hormone (hGH).Suitable therapeutics may also be examined using an animal model, forexample as described in the Examples below. Animal models arewell-developed, and fairly predictive of success in humans.

[0012] Following diagnosis of ischemic damage, an angiotensin IIinhibitor is prescribed at a dosage determined in view of the patient'scondition, weight, severity of symptoms, and the like. Angiotensin IIinhibitors include ACE inhibitors, such as captopril or enalapril, andAT₁ receptor blockers such as losartan (others, such as valrantan andirbesartan are in clinical efficacy trials), prescribed at a dosagedetermined by the patient's condition. The typical starting dose rangesfrom about 2.5 mg/day to about 20 mg/day for ACE inhibitors such asenalapril, and about 12.5 mg/day to about 50 mg/day for AT₁ inhibitorssuch as losartan. Treatment with the angiotensin II inhibitor iscontinued until substantial beneficial remodeling of the heart occurs,or until the period for unfavorable remodeling has passed. Treatment ispreferably continued until remodeling is complete (about 10 to about 12weeks), or may be continued indefinitely, particularly if heart failureis present. The patient's recovery progress may be monitored usingstandard techniques, for example echocardiography. At this point,administration of the angiotensin II inhibitor can be discontinued (asit may also inhibit the effect of the growth hormone), or relatively lowdose ACE inhibition or AT₁ blockade maintained. The selected growthhormone is thus administered until the desired effect is achieved,typically from about two to about three weeks, preferably about twoweeks. However, growth hormone was safely administered for three monthsto patients with idiopathic dilated cardiomyopathy on unknown doses ofACE inhibitors. The presently preferred growth hormone is hGH(Protropin®, Genentech), administered at a dosage of about 2 to about 6IU, preferably about 4 IU every other day. Another suitable GH isNutropin® (Genentech Inc.), administered at a dosage of about 1 to about3 μg, preferably about 2 μg every other day. Again, the patient'srecovery progress may be monitored using standard techniques. Ifdesired, the process may be repeated.

[0013] Suitable compositions or devices comprising growth hormoneinclude those suitable for controlling blood levels of growth hormone.For example, compositions or devices for the prolonged or controlleddelivery of GH over the period of time disclosed herein are useful inthe context of the present invention. A preferred means for controllingblood levels of GH is to administer the GH in the form of a polymericmatrix that releases GH as a consequence of diffusion from and/ordegradation of a polymer implant.

[0014] A variety of biodegradable and non-biodegradable polymers havebeen used for such applications, including polyesters such aspoly(lactide-co-glycolide)s, polyorthoesters and ethylenevinyl acetatepolymers. In general the delivery of the GH is controlled by theselection of the appropriate polymer, polymerization conditions, drugloading and the presence or absence of various excipients.

[0015] Particularly preferred among the compositions for prolongeddelivery of GH include bioerodible polymers such as poly(lactide),poly(lactide-co-glycolide), poly(caprolactone), polycarbonates,polyamides, polyanhydrides, polyamino acids, polyorthoesters,polyacetals, polycyanoacrylates and degradable polyurethanes andnon-erodible polymers such as polyacrylates, ethylene-vinyl acetatepolymers and other acyl substituted cellulose acetates and derivativesthereof, non-erodible polyurethanes, polystyrenes, polyvinylchloride,polyvinylfluoride, poly(vinyl imidazole), chlorosuphonated polyolefins,and polyethylene oxide.

[0016] Preferred biodegradable polymers are aliphatic polyesters, e.g.,homopolymers or copolymers synthesized from one or more kinds of-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid,2-hydroxybutyric acid, etc.), hydroxydicarboxylic acids (e.g., malicacid, etc.) and hydroxytricarboxylic acids (e.g., citric acid, etc.), ortheir mixtures, and the like. When the -hydroxycarboxylic acids arechiral compounds, they may be any of D-, L- and DL- configuration. It ispreferable that the ratio of the D-/L-configuration (mol %) is in therange of about 75/25 to about 25/75. More preferred is ahydroxycarboxylic acid wherein the ratio of the D-/L- configuration (mol%) is in the range of about 60/40 to about 30/70.

[0017] Examples of -hydroxycarboxylic acid polymers are lacticacid-glycolic acid copolymer and 2-hydroxybutyric acid-glycolic acidcopolymer. A particularly preferred -hydroxycarboxylic acid copolymer isa lactic acid-glycolic acid.

[0018] The polylactic acid preferably has a weight average molecularweight of about 1,000 to about 100,000. More preferred is a polymer acidhaving the weight average molecular weight of about 5,000 to about70,000. Particularly preferred is a polylactic acid having the weightaverage molecular weight of about 6,000 to about 15,000.

[0019] The compositional ratio (lactic acid/glycolic acid, mol %) in thelactic acid-glycolic acid copolymer is preferably about 100/0 to about40/60, more preferably about 90/10 to about 45/55, and most preferablyabout 60/40 to about 40/60. The weight average molecular weight of thelactic acid-glycolic acid copolymer is preferably about 3,000 to about20,000, and more preferably about 4,000 to about 15,000.

[0020] Preferred compositions are described in, for example WO94/12158,which disclosure is specifically incorporated herein by reference.

[0021] Growth hormone is particularly suited for complexing with variousmetal cations having a valency of 2⁺ or more. These compositions aresuitable for the methods described herein. The metal cations includepolyvalent metals such as zinc (II), iron (II, III), copper (II), tin(II, IV), and aluminum (II, III) with an inorganic or organic acid. Themetal is preferably a polyvalent metal, and more preferably an alkalineearth metal: particularly preferred metals are calcium and zinc.

[0022] Polyvalent metal salts that may be used include salts of zincwith an inorganic acid, e.g., zinc halides (for example, zinc chloride,bromide, iodide, or fluoride), zinc sulfate, zinc nitrate, zincthiocyanate, etc.; salts of zinc with an organic acid, e.g., aliphaticcarboxylic acid zinc salts (e.g., zinc carbonate, acetate, glycolate,lactate, tartrate, etc.), aromatic zinc salts (e.g., zinc benzoate,salicylate, phenolsulfonate, etc.); salts of calcium with an inorganicacid, e.g., calcium halide (e.g., calcium chloride, bromide, iodide,fluoride, etc.), calcium sulfate, nitrate, thiocyanate, etc.; salts ofcalcium with an organic acid, e.g., aliphatic carboxylic acid calciumsalt (e.g., calcium carbonate, acetate, propionate, oxalate, tartrate,lactate, citrate, gluconate, etc.) and aromatic calcium salts (e.g.,calcium benzoate, salicylate, etc.).

[0023] The preferred polyvalent metal salt includes zinc acetate andzinc carbonate. When the GH contains a metal, the molar ratio of metalcation to GH is between about 4:1′ and about 10:1 and more preferablyabout 6:1. Preferred compositions of GH comprising zinc are described inWO96/40072.

[0024] The biodegradable polymer composition can be produced byemulsifying and dispersing an aqueous solution or solid form of GH or ametal salt in an organic solvent solution of a biodegradable ornon-biodegradable polymer to prepare a water/oil (w/o) or oil/water(o/w) emulsion or an organic solution or suspension of a biodegradablepolymer containing a metal salt. The resulting substances are washed anddried or subjected to an in-water drying method, phase separationmethod, spray drying method or the like with washing and drying. Methodsfor producing a biodegradable or non-biodegradable polymer are wellknown in the art and include: In-water drying method (water/oil/water orw/o/w method), in-water drying method (o/w method), phase separationmethod (Coacervation method), and the spray drying method. Preferredamong the methods of preparation are those described in U.S. Pat. No.5,019,400 to Gombotz et al., specifically incorporated herein byreference.

[0025] The concentration of GH comprised in the sustained-releasepreparation in the present invention is, for example, about 0.1 to about30% (w/w), and preferably about 10% to about 20% (w/w).

[0026] The sustained-release preparation may be administered in the formof microcapsules or in various dosage forms such as non-oralpreparations (e.g., intramuscular-, subcutaneous- or visceral-injectable or nasal-, rectal or uterine-transmucosal preparation), ororal preparations (e.g., capsules such as hard capsule and soft capsule,solid preparations such as in granules and powder, liquid preparationssuch as suspensions).

[0027] The particularly preferred sustained-release preparation isadministered by injection. To prepare an injection using themicrocapsules obtained above, the microcapsules may be formulated with adispersant (e.g., surfactants such as Tween® 80, HCO-60; polysaccharidessuch as carboxymethylcellulose, sodium alginate, sodium hyaluronate;protamine sulfate; polyethylene glycol 400, etc.), a preservative (e.g.,methyl paraben, propyl paraben, etc.), an isotonizing agent (e.g.,sodium chloride, mannitol, sorbitol, glucose, etc.), and a localanesthetic (e.g., xylocaine hydrochloride, chlorobutanol, etc.) toprovide an aqueous suspension, or dispersed with vegetable oil (e.g.,sesame oil, corn oil, etc.), or a mixture thereof with a phospholipid(e.g., lecithin) or medium-chain fatty acid triglycerides (e.g., Migriol812) to provide an oily suspension.

[0028] When the sustained-release preparation comprises microcapsules,the microcapsules are preferably fine particles. The size ofmicrocapsules for an injectable suspension may be selected from therange satisfying the requirements for the degree of dispersion andpassage through the needle used for the injection. For example, themicrocapsule particle size may be within the range of about 0.1 to about300 μm, preferably about 1 to about 150 μm and more preferably about 2to about 100 μm.

[0029] Methods of preparing microcapsules as a sterile preparationinclude, but are not limited to, the method in which the entireproduction process is sterile, the method in which gamma rays are usedas the sterilant, and method in which an antiseptic is added during themanufacturing process.

[0030] The sustained-release preparation can be safely used in mammals(e.g., humans, bovine, swine, dogs, cats, mice, rats, rabbits, etc.).

[0031] When the sustained-release preparation is a one-week-long actionformulation, the dosage of the bioactive polypeptide can be chosen fromthe range of about 0.0001 to about 10 mg/kg body weight per an adult.The more preferred dosage can be suitably chosen from the range of about0.0005 to about 1 mg/kg body weight. The preferred administrationfrequency of the sustained-release preparation depend on the dosageform, the duration of the release, the subject animal species and otherfactors.

EXAMPLES

[0032] The following examples are set forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to practice the invention, and are not intended to limit the scopeof the invention. Efforts have been made to ensure accuracy with respectto numbers used (e.g., amounts, temperature, molecular weight, etc.),but some experimental errors and deviation should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1

[0033] Normal Rats: Normal female Sprague Dawley rats (240-260 g each)were randomized to one of the following four groups: rats receivingplacebo for 6 weeks (Control group, n=8); rats treated with placebo for6 weeks with concomitant rhGH (2 mg/kg bid) for the final 2 weeks (GHgroup, n=8); rats treated with relatively low-dose losartan (L) (20mg/kg/day in drinking water) for 6 weeks with concomitant rhGH (2mg/kg/bid, SC) during the final 2 weeks (LowL+GH, n=8); and rats treatedwith high dose L (2 g/L in drinking water) for 6 weeks with concomitantrhGH (2 mg/kg/bid, SC) during the final 2 weeks (HighL+GH, n=8). Inthese 32 animals, the responses of body weight (BW) and the weights ofthe heart and other selected organs were then examined.

[0034] MI Rats: Female Sprague-Dawley rats (230-260 g each) wereanesthetized with a mixture of ketamine hydrochloride (100 mg/kg) andxylazine (10 mg/kg) given intraperitoneally. Complete occlusion of theproximal left coronary artery was performed as described by R. Duerr etal., J Clin Invest (1995) 95:619-27; and R. L. Duerr et al., Circulation(1996) 93:2188-96. Briefly, animals were placed in the supine position,intubated and respired with a rodent ventilator. A left thoracotomy wasperformed, the pericardium opened and the left coronary artery, which isintramural, was encircled within the myocardium between the left atrialappendage and the right ventricular outflow tract with a curved needleand 5-0 silk suture. Upon tying the ligature, complete occlusion wasevidenced by a distinct regional color change of the myocardium,together with the development of acute ST segment elevations on theelectrocardiogram (ECG). The chest was then closed in layers and thepneumothorax evacuated.

[0035] Following the operation, animals were caged in proportion tosize, given water and standard rat chow ad libitum, and housed in aclimate-controlled environment subjected to 12 hour light/dark cycles.Of the 120 animals subjected to surgery, 76 (63%) survived to theseventh postoperative day and showed clear ECG and echocardiographicevidence of myocardial infarction. They were then randomly assigned toone of the following three groups: rats receiving no treatment for 12weeks (Control group, n=29); rats treated with losartan (L, 2 g/L indrinking water) for 10 weeks followed by rhGH alone (2 mg/kg, bid SC)for 2 weeks (GH[L] group, n=21); and rats treated with L for 10 weekswith placebo (given bid) for the final two weeks (P[L] group, n=26). Thelast group was included primarily to assess the degree of leftventricular remodeling and function that might persist for 2 weeks afterlosartan was discontinued, at the time that the control and GH(L) groupswere compared.

[0036] Echocardiographic examinations were performed just beforerandomization at one week, and again at 13 weeks after coronaryligation, prior to terminal hemodynamic studies. Animals wereanesthetized in the same manner as described for surgery. The chest wasshaved and rats were examined in the left lateral recumbent position.The instrument used was an annular array system (Apogee CX, ATLInterspec, Bothell, Wash.) with the transducer operating at 7.5 MHZ, ata minimum depth setting of 3 cm and sector width of 40-50 degrees tooptimize resolution and penetration for 2-D and M-mode imaging (see N.Tanaka et al., Circulation (1996) 94:1109-17). Parasternal short-axisviews of the left ventricle (LV) at the level of the papillary muscleswere obtained, and two-dimensional guided M-mode echocardiographictracings were recorded at paper speeds of 50 to 100 mm/s. Leftventricular anterior and posterior wall thickness (AWT, PWT) atend-diastole (ED) and LV internal dimensions (D) at end-systole and EDwere measured according to criteria recommended by the American Societyof Echocardiography (D. J. Sahn et al., Circulation (1978) 58:1072-83)and LV percent (%) fractional shortening (FS) was calculated.

[0037] For pulsed wave Doppler examination, the minimum sample volume(0.6 mm) was used and the pulse repetition frequency was 5 KHZ. Wemeasured the ratio of peak A velocity to peak E velocity (A/E) oftransmittal flow and the deceleration time (DT) of the E wave, which wasdefined as the time from the E wave peak to a linear extrapolation ofthe velocity signal to baseline, using a modified parasternal long-axisview (S. E. Litwin et al., Circulation (1994) 89:345-54). Also, in 30normal, age-matched female Sprague-Dawley rats M-mode and pulsed waveDoppler echocardiographic studies were performed.

[0038] Infarct size was estimated by histologic analysis of multiple LVsections and echocardiography in animals, the latter by observing theakinetic portion of the LV in the short axis view and calculating theratio of the akinetic portion of the LV endocardial circumference tothat of the entire circumference on stop-frame 2D images at ED.

[0039] Tracings were recorded on videotape, a digital color printer(Sony, EP-1800MD), or a black and white thermal printer (Sony,UP-870MD), all measurements being made in a blinded manner.

[0040] Within 3 days after the second echocardiographic study, the ratswere anesthetized, intubated, and mechanically ventilated in the supineposition as described above. Under closed-chest conditions, a 2 Frhigh-fidelity catheter-tip micromanometer (model SPR-407, Millar,Houston, Tex.) was passed into the LV via the right carotid artery tomeasure the LV pressure; its first derivative was also monitored usingan electronic differentiating circuit. Zero baseline reference for thecatheter was obtained by placing the sensor in a 32 □C water bath for atleast 30 min before insertion and after withdrawal. Because of theoccasional drift in the LV ED pressure signal with the high-fidelitycatheter, in some rats a fluid-filled catheter (PE 50) also was insertedin the LV to measure LV ED pressure. A similar fluid-filled catheter wasinserted into the left carotid artery to record the aortic pressure inall rats. Pressure data were transcribed on a chart recorder anddigitized data were recorded simultaneously at a sampling rate of2000/sec using Cordis software (Dataq Instruments, Inc., Akron, Ohio).Hemodynamic variables (LV peak and ED pressure and LV dP/dt_(max)) wereanalyzed by averaging at least 10 consecutive beats with the aid ofCordat software (Dusseldorf, Germany). The time constant of LVisovolumic pressure decay (Tau) was calculated according to a variableasymptote method (G. L. Raffet al., Circ Res (1981) 48:813-24). Theequation simplifies to P=P₀exp(−t/T)+P_(B), dP/dt=−1/T(P-P_(B)), whereP₀ is pressure and P_(B) is the intercept of pressure at time zero and Tis tau. T is determined from the inverse slope of the linear relationdP/dt versus (P-P_(B)).

[0041] Cardiac output was measured in subgroups of animals using 15 μmfluorescent labeled microspheres, as described by R. L. Duerr (1996)supra. In brief, under anesthetized conditions as described above, theright carotid artery was cannulated and a PE 50 tubing advanced into theLV. An incision was made over the right groin, and a PE 50 tubingadvanced via the femoral artery into the right common iliac artery. Themicrospheres were suspended in 10 ml of 0.1 % Tween® 80 and 0.9% saline(Molecular Probes, Inc., Eugene, Oreg.), and vials were vortexed at roomtemperature for a minimum of 20 min prior to injection to insureadequate mixing. Reference blood sampling via the femoral arterycatheter was commenced 15 to 20 seconds prior to injection and continuedfor a minimum of 30 sec after flushing the LV catheter. Approximately2.9×10⁴ spheres in 0.3 ml were injected into the LV over a 15 secondperiod using a Hamilton injection syringe, and the syringe and LVcatheter were flushed with 1 ml of normal saline for an additional 20 to30 sec. The reference sample volume was equivalent to the volume of themicrosphere suspension and saline injected (1.3 ml). Using this method,measurements of cardiac output were performed in triplicate in eachanimal. Fluorescent dye present in each sample was measured on a PerkinElmer LS50B luminescence spectrometer. The photomultiplier tube voltagewas set at 820 V and the excitation and emission slit widths were set at4 and 5 nm, respectively. A cutoff filter eliminated all light below 350um wavelength, as described by R. L. Duerr (1996) supra.

[0042] After the hemodynamic measurements, the rats were euthanized andwet weights of heart and other selected organs, including liver, spleen,and kidney, were measured. Cardiac fixation was then performed asdescribed by R. L. Duerr (1996) supra. In brief, polyethylene catheterswere introduced into the LV apex and the descending aorta. The LVchamber was filled from a reservoir and maintained at 10 mmHg. Afterwashout of blood from the coronary arteries with heparinized saline, themyocardium was perfused retrogradely from the aorta with 10% bufferedformalin at a constant pressure of 60 mmHg for 20 minutes. The heart wasthen excised and immersed in 10% formalin for 24 hours. Subsequently,the atria and adhesions were dissected away and the right ventricle (RV)and LV were separated and weighted, the interventricular septum beingincluded with the LV. The right tibia was dissected and its length fromthe condyles to the tip of the medial malleolus was measured with amicrometer caliper by the method of Yin et al., Am J Physiol (1982);243. To compare heart weights among groups, the weights were normalizedby tibial length (TL) as well as by body weight (BW).

[0043] The LV was sectioned from the apex to base in a parallel line tothe atrioventricular groove into four slices 2 to 2.5 mm in thickness,which were embedded in paraffin. Sections 10 μm thick were cut, mountedand stained with Masson's trichrome. These four slides were thenanalyzed blindly to assess infarct size, the slides being projected witha microprojector (Jena, Germany) at a magnification of ×13 andmeasurements made using computerized planimetry. The percent infarctsize was determined from the ratio of the sum of the scar lengths alongthe endocardial and epicardial surfaces to the sum of the totalendocardial and epicardial circumferences (T. E. Raya et al., Am JHypertens (1991) 4:334S-40S).

[0044] In order to confirm AT₁ receptor blockade with losartan,angiotensin II dose-response curves for changes in carotid arterialpressure were carried out in an additional 14 female Sprague-Dawley rats(260-300 g/each). Eight rats were normal, 4 untreated and 4 treated for2 weeks with losartan (2 g/L in drinking water). In 6 rats a large MIwas produced 3 to 4 weeks prior to study, of which 3 received losartan(2 g/L in drinking water) for 2 weeks and 3 were untreated. Losartantreatment for 2 weeks produced marked rightward shifts of theangiotensin II-blood pressure dose-response curves in both untreatednormal rats and in rats with chronic MI.

[0045] The last dose of GH was given 4 to 6 hours before the hemodynamicstudy. Just prior to euthanasia, 1 ml of blood was obtained in aheparinized syringe from the LV cavity and centrifuged, the plasmaobtained and stored at −70 □C for subsequent analysis. Human GH wasmeasured by a sensitive and specific ELISA assay (A. C. Celniker et al.,J Clin Endocrinol Metab (1989) 68:469-76), which does not detect rat GH.Total IGF-1 levels were measured after acid-ethanol extraction byradioimmunoassay, using human IGF-1 (Genentech M3-RD1) as the standardand a rabbit anti-IGF-1 polyclonal antiserum (R. W. Furlanetto et al., JClin Invest (1977) 60:648-57) as described by R. Yang et al.,Circulation (1995) 92:262-67; H. Jin et al., J Cardiovasc Pharmacol(1995) 26:420-25.

[0046] Among the 76 rats randomized to the experimental protocol, 10animals (6 Controls, one from the P(L) group, 3 in the GH(L) group) diedduring the treatment, and 5 (one Control, one P(L), and 3 GH(L) group)did not have clear histologic evidence of myocardial infarction orshowed a very small infarct size (<20%) despite prior ECG andechocardiographic evidence of infarction early after coronary occlusion,yielding 61 animals for analysis (22 in the Control group, 19 in theP(L) group, and 20 in the GH(L) group).

[0047] At 13 weeks, adequate echocardiographic tracings were obtained in21 control rats, 19 in the P(L) group and 20 in the GH(L) group.Hemodynamic variables were successfully measured in 20 Control rats, 17in the GH(L) group, and 19 in the P(L) group, while cardiac output wasmeasured in subgroups of 8 control rats, 10 rats in the GH(L) group, and6 in the P(L) group. Serum levels of rhGH and IGF-1 were measured in 16control rats, 16 in the GH(L) group and 15 rats in the P(L) group.

[0048] In 17 of the 61 animals, the quality of one or more of the 4histologic sections was found to be unsatisfactory for measuring infarctsize. In 36 of the rats having both 2D echocardiographic and histologicassessments of infarct size, we obtained a good correlation between themethods (y=0.46x+21.5, r=0.77, p<0.001), also interobserver variabilityof infarct size by the 2D method showed good agreement by two observers,with a mean difference of 0.86% and coefficient of variation of 10.26%.Therefore, we used histologic measurements of myocardial infarct size in44 animals, and in the 17 animals in which histological assessment wasnot feasible infarct size was estimated by 2D echocardiography.

[0049] Data are shown below expressed as mean ±SD, except for thedose-response effects of angiotensin II on blood pressure (mean ±SEM).Intergroup comparisons between controls, GH(L), and P(L) groups in therats with MI were made using an analysis of variance with post hoc testsby the Neuman-Keuls multiple range method. Two-tailed unpaired t-testswere used to compare variables in untreated control MI rats with thosein normal rats (different numbers of rats having each measurement), andthe groups of normal rats were compared using two-tailed t-tests withBonferonni corrections. All data were analyzed in a blinded fashion. Aprobability value of P<0.05 was accepted as statistically significant.

[0050] Results:

[0051] The BW and weights of selected organs were significantlyincreased in the GH, LowL+GH and HighL+GH groups compared with those inthe control group, without significant differences among the 3 groupsreceiving GH (Table 1). TABLE 1 Body, organ and cardiac weights innormal rats after AT₁ blockade and GH Control GH Low L + GH High L + GHNumber 8 8 8 8 BW (g) 273.7 ± 17.6 319.9 ± 15.9* 310.8 ± 17.5* 328.4 ±26.7* Liver (g) 10.20 ± 1.25 13.69 ± 0.94* 13.77 ± 1.17* 14.18 ± 1.94*Spleen (g) 0.77 ± 0.10 1.21 ± 0.10* 1.10 ± 0.19* 1.09 ± 0.17* Kidney (g)1.99 ± 0.13 2.30 ± 0.19* 2.42 ± 0.19* 2.34 ± 0.11* TL (cm) 3.96 ± 0.064.05 ± 0.08 3.96 ± 0.07 3.99 ± 0.06 HW (g) 1.31 ± 0.10 1.42 ± 0.12* 1.40± 0.13 1.22 ± 0.06** HW/BW (%) 0.48 ± 0.03 0.45 ± 0.04 0.45 ± 0.03 0.37± 0.04*,** HW/TL (%) 33.0 ± 2.6 35.1 ± 2.5* 35.3 ± 3.3 30.6 ± 1.9** LVwt (g) 0.96 ± 0.10 1.04 ± 0.06 1.02 ± 0.07 0.92 ± 0.08** LV/BW (%) 0.35± 0.03 0.33 ± 0.01 0.33 ± 0.02 0.28 ± 0.04* LV/TL (%) 24.2 ± 2.5 25.7 ±1.6 25.8 ± 1.8 23.2 ± 2.1** RV wt (g) 0.23 ± 0.05 0.26 ± 0.02 0.25 ±0.04 0.24 ± 0.05 RV/BW (%) 0.08 ± 0.02 0.08 ± 0.01 0.08 ± 0.01 0.07 ±0.01 RY/TL (%) 5.8 ± 1.4 6.3 ± 0.6 6.2 ± 1.0 6.0 ± 1.2

[0052] In the HighL+GH group, the heart weight, absolute and normalizedto the BW and TL, were significantly decreased compared to the GH group,and the LV weights, absolute and normalized to TL also weresignificantly decreased (Table 1). The heart weights and LV weights,absolute and normalized to the BW and to the TL, were not significantlydifferent between the GH and LowL+GH groups. The RV weights, absoluteand normalized to the BW and to the TL were not significantly differentamong all 4 groups. In the GH group, absolute heart weights, and heartweight normalized to TL (but not to BW) were significantly increasedcompared to those in the control group.

[0053] These data provide evidence that in the normal rat, thehypertrophic cardiac effect of GH is blocked by high dose but not byrelatively low dose losartan.

[0054] Myocardial infarct size in all 61 rats averaged 42.6±7.7% of theLV circumference at 13 weeks after coronary artery ligation, and therewere no significant differences in the average infarct size amongcontrols and the two treatment groups (Table 2).

[0055] In the GH(L) group, the average serum rhGH level obtained earlyafter GH injection was markedly increased, and the average IGF-1 levelwas significantly elevated (by 34%) compared to control rats withinfarction.

[0056] Treatment for 2 weeks with GH following L (the GH(L) group)caused significant increases in the body weight (BW) (13% compared tocontrols), and in the weights of selected organs including the liver,spleen, and kidney, compared with those in controls and to the grouptreated with placebo for 2 weeks after L (the P(L) group). The BW andorgan weights did not differ significantly between the control and P(L)groups.

[0057] Heart and ventricular weights, absolute and normalized to the BWand to the TL, are summarized in Table 2. The LV weight/BW ratio wassignificantly reduced compared to controls in both the GH(L) and theP(L) groups, whereas the reductions in heart weight/BW were notsignificant; the LV weight normalized to the TL was reduced in the P(L)group compared to controls. TABLE 2 Myocardial Infarct size, HormoneLevels, and Body, Organ, Cardiac weights in rats with MyocardialInfarction Control P(L) GH(L) Number 22 19 20 infarct size (%) 41.6 ±7.6 42.3 ± 7.5 41.1 ± 8.0 GH (ng/ml) 0.9 ± 0.2 0.9 ± 0.2 16684 ± 1250*+(n = 16) (n = 15) (n = 16) IGF-1 (ng/ml) 298.6 ± 80.5 338.5 ± 64.3 399.2± 127.1* (n = 16) (n = 15) (n = 16) BW (g) 278.6 ± 18.1 277.7 ± 18.8314.8 ± 33.3*+ Liver (g) 9.36 ± 1.44 8.91 ± 1.12 12.72 ± 1.44*+ Spleen(g) 0.93 ± 0.18 0.95 ± 0.16 1.38 ± 0.33*+ Kidney (g) 1.94 ± 0.23 1.99 ±0.20 2.23 ± 0.19*+ TL (cm) 4.02 ± 0.10 4.03 ± 0.09 4.03 ± 0.10 HW (g)1.70 ± 0.41 1.47 ± 0.25 1.62 ± 0.37 HW/BW (%) 0.61 ± 0.16 0.53 ± 0.090.51 ± 0.12 HW/TL (%) 42.4 ± 10.2 36.4 ± 6.3 40.3 ± 9.1 LV wt (g) 0.98 ±0.17 0.87 ± 0.10 0.95 ± 0.17 LV wt/BW (%) 0.35 ± 0.06 0.32 ± 0.04* 0.30± 0.06* LV wt/TL (%) 24.5 ± 4.1 21.7 ± 2.5* 23.5 ± 4.4 RV wt (g) 0.26 ±0.12 0.21 ± 0.04 0.29 ± 0.12+ RV wt/BW (%) 0.10 ± 0.05 0.08 ± 0.01 0.09± 0.04 RV wt/TL(%) 6.6 ± 3.1 5.2 ± 0.9 7.2 ± 3.0+ # = weight. Heartweight was measured before fixation, LV and RV weights were measuredafter fixation.

[0058] The RV weight, absolute and normalized to the TL, was increasedsignificantly in the GH(L) group compared to that in the P(L) group, andwas not significantly different from controls.

[0059] Heart rate and LV systolic and mean aortic pressures were closelysimilar among the three groups (Table 3). LV end-diastolic pressure(EDP) was elevated in controls (11.9 vs 3.7 mmHg in 17 normal rats,P<0.01) (Table 4). The reduction in LVEDP was not significant in theGH(L) group compared with controls but it was significantly reduced inthe P(L) group (Table 3). LV dP/dt_(max) was reduced in controls (4699vs 6545 mmHg/s in 17 normal rats, p<0.001)(Table 4). LV dP/dt_(max) wassignificantly greater in the GH(L) group than in controls, but it wasnot significantly increased in the P(L) group (Table 3). Tau wasshortened compared to controls in both the GH(L) and P(L) groups (Table3). TABLE 3 Hemodynamic Characteristics in Rats with MyocardialInfarction Control P(L) GH(L) Pressures: Number 20 19 17 HR (bpm) 244.8± 36.7 242.3 ± 47.5 250.9 ± 24.1 LVEDP (mmHg) 11.9 ± 7.7 3.9 ± 2.1* 8.5± 7.7 (n = 16) (n = 10) (n = 11) LVSP (mmHg) 93.1 ± 15.6 93.8 ± 18.195.6 ± 16.1 Mean AoP (mmHg) 78.0 ± 11.5 75.0 ± 13.2 79.3 ± 13.7 LvdP/dt_(max) (mmHg/s) 4699 ± 1004 5045 ± 667 5579 ± 1063* Tau (ms) 34.8 ±7.5 25.7 ± 8.1* 22.1 ± 7.9* (n = 15) (n = 19) (n = 16) Cardiac Output:Number 8 6 10 CO (ml/min) 40.6 ± 7.3 47.9 ± 5.3 58.0 ± 11.9*+ CI(ml/min/kg) 148.5 ± 24.9 178.5 ± 18.9* 189.0 ± 31.0* SV (ml/beat) 0.18 ±0.03 0.20 ± 0.02 0.25 ± 0.04*+ SVI (ml/beat/kg) 0.65 ± 0.11 0.73 ± 0.070.81 ± 0.10* Mean AoP (mmHg) 76.8 ± 13.7 76.4 ± 12.0 66.7 ± 10.8 SVR(mmHg/ml/min) 1.92 ± 0.39 1.61 ± 0.31 1.17 ± 0.18*

[0060] The cardiac index was reduced in controls (148.5 vs. 214.7ml/min/kg in normal rats, p<0.05, Table 4). The cardiac output and thestroke volume were significantly increased in the GH(L) group comparedto controls and the P(L) group. The CI was significantly higher in boththe GH(L) and P(L) groups than in controls, but the systemic vascularresistance was significantly reduced (by 39%) only in the GH(L) group(Table 3). TABLE 4 Hemodynamic and Echo findings in normal rats and ratswith myocardial infarction Normal MI (control) p value Pressures: LVEDP(mmHg) 3.7 ± 2.2 11.9 ± 7.7 <0.01 (n = 17) (n = 16) LVSP (mmHg) 105.2 ±21.0 93.1 ± 15.6 p = 0.052 (n = 17) (n = 20) Mean AoP (mmHg) 89.3 ± 17.778.0 ± 11.5 p < 0.05 (n = 17) (n = 20) LV dP/dt_(max) (mmHg/s) 6545 ±943 4699 ± 1004 p < 0.001 (n = 17) (n = 20) Tau (ms) 13.6 ± 1.8 34.8 ±7.5 p < 0.001 (n = 14) (n = 15) CI (ml/min/kg) 214.7 ± 55.4 148.5 ± 24.9p < 0.05 (n = 5) (n = 8) SVR (mmHg/ml/min) 1.44 ± 0.46 1.92 ± 0.39 p<0.05 (n = 5) (n = 8) Echocardio-graphic Data: EDD (mm) 6.08 ± 0.54 9.97± 1.12 p < 0.001 (n = 30) (n = 21) FS (%) 41.0 ± 6.3 15.4 ± 6.0 p <0.001 (n = 30) (n = 21) DT (ms) 57.6 ± 16.7 38.3 ± 7.6 p < 0.001 (n =30) (n = 21) # diastolic dimension, FS = fractional shortening of leftventricle, DT = deceleration time of E wave of transmitral flowvelocity. Numbers are mean ± SD.

[0061] The EDD of the LV was significantly increased in controls (9.97vs 6.08 mm in normal rats, p<0.001), and the % FS was reduced (15.4% vs.41.0% in normal rats, p<0.001, Table 4). The absolute EDDs were notsignificantly different in the 3 groups, but the EDD/BW was reduced inthe GH(L) group versus the control and P(L) groups (Table 5). In theGH(L) group, the % FS of the LV was significantly greater than incontrols. The anterior wall thickness (AWT) (the infarcted wall) wasabnormally thin and similar among the groups, whereas the non-infarctedposterior wall thickness (PWT) was significantly greater in the GH(L)group than in the other two groups, resulting in a significant decreasein the EDD/PWT ratio compared with the control and P(L) groups (table5).

[0062] The A/E ratio of Doppler-determined mitral flow velocity tendedto be lower in the control group compared with the other groups, but thedifferences were not significant (table 4). The deceleration time (DT)of the E wave was shortened in controls (38.3 vs. 57.6 ms in 30 normalrats, p<0.001). The DT was significantly prolonged in both the GH(L) andP(L) groups compared to controls (Table 5). TABLE 5 M-Mode and DopplerEchocardiographic Findings in Rats with Myocardial Infarction ControlP(L) GH(L) Number 21 19 20 EDD (mm) 9.97 ± 1.12 9.52 ± 1.16 9.91 ± 0.42EDD/TL 0.25 ± 0.03 0.24 ± 0.03 0.25 ± 0.01 EDD/BW (mm/g) 0.036 ± 0.0050.035 ± 0.004 0.032 ± 0.004*+ EDD/PWT 7.97 ± 1.83 7.29 ± 1.28 6.76 ±0.99* ESD (mm) 8.47 ± 1.36 7.86 ± 1.20 7.90 ± 0.72 FS (%) 15.4 ± 6.017.7 ± 5.3 20.3 ± 5.6* AWT (mm) 0.81 ± 0.15 0.80 ± 0.19 0.79 ± 0.15 PWT(mm) 1.28 ± 0.17 1.33 ± 0.16 1.49 ± 0.19*+ A/E ratio 0.40 ± 0.27 0.53 ±0.15 0.45 ± 0.29 DT (ms) 38.3 ± 7.6 44.7 ± 5.6* 48.5 ± 9.8*

[0063] In this study, experimental LV failure at approximately 3 monthsafter coronary ligation in untreated rats with myocardial infarctionsthat involved more than 20% of the LV circumference was characterized bya reduced cardiac index, markedly increased LV cavity dimension,increased LVEDP, severely reduced LV fractional shortening and LVdP/dt_(max), and impairment of LV relaxation (tau) and early filling,when compared to normal rats.

[0064] The data demonstrates that after favorable LV remodeling had beeninduced by AT₁ receptor blockade for 10 weeks, GH administration alonefor 2 weeks was associated with (1) improved stroke volume and cardiacindex, (2) decreased system vascular resistance, (3) increased LVfractional shortening, (4) modest enhancement of LV myocardialcontractility (LV dP/dt_(max)), (5) a hypertrophic effect on the LVwhich contributed to an improved ratio of LV diastolic dimension to wallthickness, and (6) improved LV relaxation (tau) and early diastolicfilling rate. Further, some of these effects, particularly those ondiastolic function and the cardiac index, undoubtedly received acontribution from a persistent favorable action of AT₁ blockade, evidentin rats in which losartan was replaced by placebo for 2 weeks.

[0065] The instant invention is shown and described herein at what isconsidered to be the most practical and preferred embodiments. It isrecognized, however, that departures may be made therefrom which arewithin the scope of the invention and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

What is claimed:
 1. A method for treating heart failure after myocardialinfarction in a subject, comprising: a) administering an angiotensin II(AT₁) receptor blocker to said subject for a period beginning about thetime of myocardial infarction to about ten weeks; and b) administeringgrowth hormone beginning about ten weeks after myocardial infarction. 2.The method of claim 1, wherein angiotensin II (AT₁) receptor blockeradministration is discontinued about 10 weeks after myocardialinfarction.
 3. The method of claim 1, wherein said AT₁ receptor blockercomprises losartan.
 4. A method of treating heart failure in a subjectfollowing an ischemic event, comprising; a) administering an angiotensinII inhibitor to said subject over a period beginning about the time ofsaid ischemic event, and continuing for a period sufficient tosubstantially permit favorable left ventricular remodeling; and b)administering a growth hormone to said subject at a time approximatelyafter said ventricular remodeling period.
 5. The method of claim 4,wherein administration of said angiotensin II inhibitor is discontinuedat about the time growth hormone administration begins.
 6. The method ofclaim 4, wherein said angiotensin II inhibitor comprises an AT₁ receptorinhibitor.
 7. The method of claim 6, wherein said AT₁ receptor inhibitorcomprises losartan.
 8. The method of claim 4, wherein said growthhormone is human growth hormone.
 9. The method of claim 4, wherein saidangiotensin II inhibitor is administered beginning within seven days ofsaid ischemic event.
 10. The method of claim 9, wherein said angiotensinII inhibitor is administered for about 8 to about 12 weeks.
 11. Themethod of claim 10, wherein said angiotensin II inhibitor isadministered for about 10 weeks.
 12. The method of claim 9, wherein saidgrowth hormone is administered for about one to about three weeks. 13.The method of claim 4, wherein said growth hormone is administered forabout two weeks.
 14. The method of claim 4, wherein said angiotensin IIinhibitor is administered at a reduced dosage after said ventricularremodeling period.
 15. The method of claim 14, wherein said reduceddosage is less than about one half the dosage prior to the end of saidventricular remodeling period.
 16. The method of claim 4, wherein stepsa) and b) are repeated.